Method for producing steviol and steviol glycoside using aobgl1 homolog

ABSTRACT

The present invention provides a method for producing a steviol glycoside and/or steviol, said method including a step in which a steviol glycoside having at least one unbranched β1,2-glycosidic bond is reacted with the glycosidase AOBGL1 and/or AOBGL3, or a variant thereof, so as to cleave the β1,2-glycosidic bond.

TECHNICAL FIELD

The present invention relates to a method for producing a steviolglycoside and steviol.

BACKGROUND ART

The leaves of Stevia rebaudiana of the Asteraceae family contain asecondary metabolite called “steviol” which is a kind of diterpenoid.Steviol glycosides, which are products of the addition of sugars tosteviol, include those having sweetness about 300 times higher than thatof table sugar. Such steviol glycosides are used as non-caloricsweeteners in the food industry. Obesity is becoming more of a serioussocial issue on an international scale, and non-caloric sweeteners areincreasingly demanded from the viewpoint of promotion of health andreduction of medical cost. Currently, aspartame and acesulfamepotassium, which are artificially-synthesized amino acid derivatives,are used as artificial sweeteners. However, naturally-occurringnon-caloric sweeteners such as steviol glycosides are expected to besafer and gain more public acceptance.

Among steviol glycosides, stevioside is a compound in which threeglucose units are added to steviol, and is contained in the largestamount in the leaves of common Stevia rebaudiana. Stevioside has adegree of sweetness about 300 times higher than that of sucrose, but hasslightly bitter taste. Rebaudioside A, which is another steviolglycoside, is a compound in which four glucose units are added tosteviol, and has a degree of sweetness about 400 times higher than thatof sucrose. Stevioside and rebaudioside A are primary substancesresponsible for the sweetness of Stevia rebaudiana. There are also knownglycosides such as rebaudioside D in which five glucose units are addedto steviol and rebaudioside M in which six glucose units are added tosteviol. It is also known that Rubus suavissimus contains rubusoside inwhich one glucose unit is added at each of the 13 and 19 positions ofsteviol and that this rubusoside is a primary sweet component of Rubussuavissimus. In addition to the above glycosides, glycosides consideredto be reaction intermediates and analogs differing in the type of sugarare known to exist (FIG. 1).

Meanwhile, steviol is known to have, for example, improving effect oncognitive function.

If an enzyme acting only on a specific glycoside bond in steviolglycosides can be used, production of a specific glycoside orelimination of an unnecessary glycoside will become possible. This willbring a lot of merits such as facilitating the improvement in taste ofStevia rebaudiana extracts or the purification of a specific steviolglycoside.

An enzyme activity to hydrolyze steviol glycosides has been reported tobe observed in some organism species. In particular, concerning theproduction of steviol glycoside-hydrolyzing enzymes by filamentous fungiof the genus Aspergillus, it has been reported that raw soy sauce has anactivity to hydrolyze stevioside into rubusoside (Non PatentLiterature 1) and that a pectinase enzyme agent, hesperidinase enzymeagent, and takadiastase enzyme agent have an activity to hydrolyzestevioside into steviol (Non Patent Literatures 2 to 4). A method hasalso been reported in which steviol is produced from stevioside by thecombined use of a pectinase enzyme agent derived from filamentous fungiof the genus Aspergillus and an enzyme agent derived from Helix pomatia(Patent Literature 1). Viscozyme L (novozyme), an enzyme agent derivedfrom Aspergillus aculeatus, has been described to have an activity tohydrolyze stevioside into rubusoside and then into steviol monoglycosylester (Non Patent Literature 5). Additionally, an extract obtained fromAspergillus aculeatus by solid culture has been described to have anactivity to convert stevioside into steviol (Non Patent Literature 6).

It has been reported that the β-glucosidase of the glycoside hydrolase(GH) family 3 encoded by the AO090009000356 gene of koji mold hydrolyzesdisaccharides with a β-glucoside bond (Non Patent Literature 7).Specifically, its specificity for hydrolysis is the highest forlaminaribiose with a β-1,3 linkage, followed by β-gentiobiose with aβ-1,6 linkage, cellobiose with a β-1,4 linkage, and sophorose with aβ-1,2 linkage. However, there has been no report on whether theβ-glucosidase has an activity to hydrolyze terpene glycosides typifiedby steviol glycosides.

Some other organisms have also been reported to have an activity tohydrolyze steviol glycosides. For example, it has been disclosed thatbacteria of the genus Clavibacter have an enzyme that decomposes theglucosyl ester bond at the 19 position of rubusoside but does notdecompose the glucoside bond at the 13 position (Patent Literature 2).Additionally, it has been reported that Flavobacteriumjohnsoniae-derived β-glucosidase has an activity to decompose steviolglycosides (an activity to hydrolyze the β-glucoside bond at the 13position and the glucosyl ester bond at the 19 position).

Although these have been found to have an activity to hydrolyze steviolglycosides, the gene responsible for this activity has not beenidentified.

Moreover, koji mold contains a large number of genes considered toencode GH3 family or GH5 family enzymes having β-glucosidase-likeactivity, and thus, even if an enzyme activity can be detected, it isnot easy to determine which gene is responsible for the activity.

CITATION LIST Patent Literature

-   Patent Literature 1: National Publication of International Patent    Application No. 2013-516963-   Patent Literature 2: Japanese Patent Laid-Open No. 10-276775-   Patent Literature 3: Japanese Patent Laid-Open No. 10-276775

Non Patent Literature

-   Non Patent Literature 1: Journal of the Japanese Society for Food    Science and Technology, vol. 37, No. 5, 369-374 (1990)-   Non Patent Literature 2: Phytochemistry, 6, 1107 (1967)-   Non Patent Literature 3: Journal of the Pharmaceutical Society of    Japan, 95, 1507 (1975)-   Non Patent Literature 4: Journal of the Chemical Society of Japan,    1981, 726 (1981)-   Non Patent Literature 5: J. Agric. Food Chem., 60, 6210-6216 (2012)-   Non Patent Literature 6: Wei Sheng Wu Xue Bao, 54(1), 62-68 (2014)-   Non Patent Literature 7: Biochim Biophys Acta., 1764 972-978 (2006)

SUMMARY OF INVENTION Technical Problem

Under such circumstances, there is a need for a novel method forproducing Steviol glycosides and Steviol.

Solution to Problem

The present inventors conducted extensive research to solve theaforementioned problem, and found that a koji mold-derived glycosidehydrolase homolog protein encoded by AOBGL1 gene has an activity tohydrolyze steviol glycosides. That is, the present inventors have foundthat the protein has an activity to specifically cleave an unbranchedβ-1,2 bond of a steviol glycoside, thus completing the presentinvention. Additionally, the present inventors have succeeded inproducing a steviol glycoside and/or steviol by further expressinganother koji mold-derived glycosidase homolog protein encoded by AOBGL3gene, thus completing the present invention.

In summary, the present invention is as set forth below.

[1]

A method for producing a steviol glycoside having no unbranchedβ-1,2-glucoside bond comprising reacting a protein selected from thegroup consisting of proteins (a) to (c) shown below with a steviolglycoside having at least one unbranched β-1,2-glucoside bond, therebycleaving said unbranched β-1,2-glucoside bond:

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or4;

(b) a protein consisting of an amino acid sequence wherein 1 to 77 aminoacids have been deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 3 or 4, and having an activity tocleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(c) a protein having an amino acid sequence having 90% or more sequenceidentity to the amino acid sequence of SEQ ID NO: 3 or 4, and having anactivity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside.

[2]

The method according to [1] above, wherein the protein selected from thegroup consisting of the proteins (a) to (c) further comprises asecretory signal peptide.

[3]

The method according to [1] above, wherein the secretory signal peptideis a peptide consisting of the amino acid sequence set forth in any oneof SEQ ID NOS: 28, 30, 32, 34, and 36.

[4]

The method according to [3] above, wherein the steviol glycoside havingat least one unbranched β-1,2-glucoside bond is selected fromrebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E,stevioside, and steviolbioside.

[5]

The method according to [4] above, wherein the steviol glycoside havingno unbranched β-1,2-glucoside bond is selected from rubusoside,rebaudioside A, and steviolmonoside.

[6]A method for producing a steviol glycoside having no unbranchedβ-1,2-glucoside bond comprising culturing a non-human transformantobtained by introducing a polynucleotide selected from the groupconsisting of polynucleotides (a) to (e) shown below into a hostproducing a steviol glycoside having at least one unbranchedβ-1,2-glucoside bond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1;

(b) a polynucleotide encoding a protein consisting of the amino acidsequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acidsequence wherein 1 to 77 amino acids have been deleted, substituted,inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4,and having an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequencehaving 90% or more sequence identity to the amino acid sequence of SEQID NO: 3 or 4, and having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditionsto a polynucleotide consisting of a nucleotide sequence complementary toa polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1and which encodes a protein having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside.

[7]

The method according to [6] above, wherein the polynucleotide selectedfrom the group consisting of the polynucleotides (a) to (e) furthercomprises a polynucleotide consisting of a nucleotide sequence encodinga secretory signal peptide.

[8]

The method according to [7] above, wherein the polynucleotide consistingof a nucleotide sequence encoding a secretory signal peptide is apolynucleotide consisting of the nucleotide sequence set forth in anyone of SEQ ID NOS: 27, 29, 31, 33, and 35.

[9]

The method according to any one of [6] to [8] above, wherein thepolynucleotide is inserted into an expression vector.

[10]

The method according to any one of [6] to [9] above, wherein thetransformant is transformed koji mold, transformed yeast, or atransformed plant.

[11]

A method for producing a steviol glycoside having no unbranchedβ-1,2-glucoside bond comprising contacting an enzyme agent derived froma non-human transformed cell obtained by introducing, into a host cell,a polynucleotide selected from the group consisting of polynucleotides(a) to (e) shown below, with a steviol glycoside having at least oneunbranched β-1,2-glucoside bond, thereby cleaving said β-1,2-glucosidebond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1;

(b) a polynucleotide encoding a protein consisting of the amino acidsequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acidsequence wherein 1 to 77 amino acids have been deleted, substituted,inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4,and having an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequencehaving 90% or more sequence identity to the amino acid sequence of SEQID NO: 3 or 4, and having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditionsto a polynucleotide consisting of a nucleotide sequence complementary toa polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1and which encodes a protein having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside.

[12]

The method according to [11] above, wherein the polynucleotide selectedfrom the group consisting of the polynucleotides (a) to (e) furthercomprises a polynucleotide consisting of a nucleotide sequence encodinga secretory signal peptide.

[13]

The method according to [12] above, wherein the polynucleotideconsisting of a nucleotide sequence encoding a secretory signal peptideis a polynucleotide consisting of the nucleotide sequence set forth inany one of SEQ ID NOS: 27, 29, 31, 33, and 35.

[14]

The method according to any one of [11] to [13]above, wherein thepolynucleotide is inserted into an expression vector.

[15]

The method according to any one of [11] to [14] above, wherein thetransformed cell is transformed koji mold, a transformed bacterium, ortransformed yeast.

[16]

The method according to any one of [11] to [15] above, wherein thesteviol glycoside having at least one unbranched β-1,2-glucoside bond isselected from rebaudioside D, rebaudioside E, stevioside, andsteviolbioside.

[17]

The method according to [16] above, wherein the steviol glycoside havingno unbranched β-1,2-glucoside bond is selected from rubusoside,rebaudioside A, stevioside, and steviolmonoside.

[18]

A method for producing at least one of rebaudioside B and steviolcomprising reacting a protein selected from the group consisting ofproteins (a) to (c) shown below, a protein selected from the groupconsisting of proteins (d) to (f) shown below, and a steviol glycosidehaving at least one unbranched β-1,2-glucoside bond:

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or4;

(b) a protein consisting of an amino acid sequence wherein 1 to 77 aminoacids have been deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 3 or 4, and having an activity tocleave an unbranched β-1,2-glucoside bond of a steviol glycoside;

(c) a protein having an amino acid sequence having 90% or more sequenceidentity to the amino acid sequence of SEQ ID NO: 3 or 4, and having anactivity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside;

(d) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or8;

(e) a protein consisting of an amino acid sequence wherein 1 to 77 aminoacids have been deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 7 or 8, and having an activity tocleave a monoglucoside bond and/or monoglucosyl ester bond of a steviolglycoside; and

(f) a protein having an amino acid sequence having 90% or more sequenceidentity to the amino acid sequence of SEQ ID NO: 7 or 8, and having anactivity to cleave a monoglucoside bond and/or monoglucosyl ester bondof a steviol glycoside.

[19]

The method according to [18] above, wherein the steviol glycoside havingat least one unbranched β-1,2-glucoside bond comprises one or moresteviol glycosides selected from rebaudioside A, rebaudioside D,stevioside, rubusoside, steviolmonoside, and steviol monoglucosyl ester.

Advantageous Effects of Invention

According to the present invention, there is provided a novel method forproducing steviol and a steviol glycoside. With the method according tothe present invention, steviol can be produced. Additionally, a steviolglycoside in which a branched trisaccharide (β-1,2 or β-1,3) is added atthe 13 position and/or 19 position of steviol can be efficientlyextracted and purified.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of various steviol glycosides and thebiosynthetic pathway from Stevia rebaudiana.

FIG. 2 shows amino acid sequences and nucleotide sequences of secretorysignals, wherein (A) shows MF(ALPHA)1 (YPL187W), (B) shows PHO5(YBR093C), and (C) shows SUC2 (YIL162W).

DESCRIPTION OF EMBODIMENTS

The present invention will be hereinafter described in detail. Thefollowing embodiments are illustrative of the present invention, and arenot intended to limit the present invention. The present invention canbe carried out in various manners, without departing from the gist ofthe invention.

Note that all documents, as well as laid-open application publications,patent application publications, and other patent documents cited hereinshall be incorporated herein by reference. The present specificationincorporates the contents of the specification and the drawings ofJapanese Patent Application No. 2015-214255, filed on Oct. 30, 2015,from which the present application claims priority.

“AOBGL1” designates a koji mold-derived β-glucosidase; the cDNAsequence, the genomic DNA sequence, the amino acid sequence, and theamino acid sequence of the mature protein are shown in SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

“AOBGL3” designates a koji mold-derived β-glucosidase; the cDNAsequence, the genomic DNA sequence, the amino acid sequence, and theamino acid sequence of the mature protein are shown in SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

1. Method for Producing a Steviol Glycoside

The present invention provides a method for producing a steviolglycoside comprising reacting a protein selected from the groupconsisting of proteins (a) to (c) shown below (hereinafter referred toas “the protein of the present invention”) with a steviol glycosidehaving at least one unbranched β-1,2-glucoside bond, thereby cleavingsaid unbranched β-1,2-glucoside bond.

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or4;

(b) a protein consisting of an amino acid sequence wherein 1 to 77 aminoacids have been deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 3 or 4, and having an activity tocleave an unbranched β-1,2-glucoside bond of a steviol glycoside; and

(c) a protein having an amino acid sequence having 90% or more sequenceidentity to the amino acid sequence of SEQ ID NO: 3 or 4, and having anactivity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside.

While the protein shown in (b) or (c) above is typically a variant of aprotein consisting of the amino acid sequence of SEQ ID NO: 3 or 4,these proteins also include proteins that can be artificially obtainedusing site-directed mutagenesis as described in, for example, “Sambrook& Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold SpringHarbor Laboratory Press 2012”, “Ausubel, Current Protocols in MolecularBiology, John Wiley & Sons 1987-1997”, “Nuc. Acids. Res., 10, 6487(1982)”, “Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)”, “Gene, 34, 315(1985)”, “Nuc. Acids. Res., 13, 4431 (1985)”, and “Proc. Natl. Acad.Sci. USA, 82, 488 (1985)”.

Examples of the “protein consisting of an amino acid sequence wherein 1to 77 amino acids have been deleted, substituted, inserted, and/or addedin the amino acid sequence of SEQ ID NO: 3 or 4, and having an activityto cleave an unbranched β-1,2-glucoside bond of a steviol glycoside”include a protein consisting of an amino acid sequence wherein, forexample, 1 to 77, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 55, 1 to 50,1 to 49, 1 to 48, 1 to 47, 1 to 46, 1 to 45, 1 to 44, 1 to 43, 1 to 42,1 to 41, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34,1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26,1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18,1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10,1 to 9 (one to several), 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3,1 or 2, or 1 amino acid residue has been deleted, substituted, inserted,and/or added in the amino acid sequence of SEQ ID NO: 3 or 4, and havingan activity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside. In general, the number of deleted, substituted, inserted,and/or added amino acid residues is preferably smaller.

Examples of such proteins include a protein having an amino acidsequence sharing 90% or more, 91% or more, 92% or more, 93% or more, 94%or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% ormore, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5%or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or moresequence identity with the amino acid sequence of SEQ ID NO: 3 or 4, andhaving an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside. In general, the value of sequence identity ispreferably greater.

As used herein, the phrase “activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside” refers to the activity tocleave an unbranched β-1,2-glucoside bond formed between two glucoseresidues in a steviol glycoside (a glycoside wherein glucose is linkedto the aglycone, steviol). As used herein, the phrase “unbranchedβ-1,2-glucoside bond” refers to a β-1,2-glucoside bond wherein theβ-1,2-glucoside-bonded glucose residues have no branched structure suchas a β-1,3-glucoside bond (“Glc-β1,2-Glu” in Table 1 below). Bycontrast, when the β-1,2-glucoside-bonded glucose residues further havea branched structure such as a β-1,3-glucoside bond, the β-1,2-glucosidebond can be referred to as a branched β-1,2-glucoside bond(“Glc-β1,2-Glu (β1,3-Glu)” in Table 1 below).

The activity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside can be confirmed by reacting the protein of the presentinvention with a steviol glycoside having at least one unbranchedβ-1,2-glucoside bond, such as stevioside, purifying the resultingreaction product, and analyzing the purified product using a knowntechnique such as liquid chromatography (LC).

The activity to hydrolyze a β-glucoside bond can be detected by using amedium containing X-β-Glc to culture a transformant genetically modifiedto express the protein of the present invention and confirming whetherthe cells and surrounding regions are stained blue. When X-β-Glc ishydrolyzed in the medium, the cells and surrounding regions are stainedblue; thus, having blue color is considered to indicate the presence ofthe activity to hydrolyze a β-glucoside bond.

Alternatively, the activity to hydrolyze a β-glucoside bond can beexamined by using p-nitrophenyl β-D-glucopyranoside as a substrate tomeasure the amount of p-nitrophenol produced by hydrolysis in terms ofabsorbance (A405).

The phrase “an amino acid sequence wherein 1 to 77 amino acid residueshave been deleted, substituted, inserted, and/or added in the amino acidsequence of SEQ ID NO: 3 or 4” means that 1 to 77 amino acid residueshave been deleted, substituted, inserted, and/or added at any 1 to 77positions in the same sequence, wherein two or more of deletion,substitution, insertion, and addition may occur simultaneously.

Examples of amino acid residues that are interchangeable are shownbelow. The amino acid residues included in the same group areinterchangeable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine,t-butylalanine, and cyclohexylalanine;

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid, and 2-aminosuberic acid;

Group C: asparagine and glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, and2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline, and 4-hydroxyproline;

Group F: serine, threonine, and homoserine; and

Group G: phenylalanine and tyrosine.

The protein of the present invention in some embodiments does notcontain a secretory signal peptide, because the secretory signal peptideis cleaved. Some other proteins of the present invention may furthercontain a secretory signal peptide, because the secretory signal peptideremains uncleaved. When the protein of the present invention contains asecretory signal peptide, it preferably contains the secretory signalpeptide at its N-terminus. The secretory signal peptide refers to apeptide domain that serves to cause extracellular secretion of a proteinbound to the secretory signal peptide. Amino acid sequences of suchsecretory signal peptides and polynucleotide sequences encoding suchamino acid sequences have been well known and reported in the art.

The protein of the present invention can be obtained by, for example,expressing a polynucleotide encoding this protein (see “thepolynucleotide of the present invention” described below) in appropriatehost cells, although it can also be produced by a chemical synthesismethod such as the Fmoc method (fluorenylmethyloxycarbonyl method) orthe tBoc method (t-butyloxycarbonyl method). The protein of the presentinvention can also be chemically synthesized using a peptide synthesizerfrom AAPPTec LLC, Perkin Elmer Inc., Protein Technologies Inc.,PerSeptive Biosystems, Applied Biosystems, or SHIMADZU CORPORATION, forexample.

As used herein, the term “steviol glycoside” refers to a glycosidewherein glucose is linked to the aglycone, steviol. Examples of stevioland steviol glycosides are represented by the following formula (I).

TABLE 1 Compound Name R₁ R₂ Rebaudioside D Glc-β1,2-Glu (β1,3-Glu)Glc-β1,2-Glu Rebaudioside E Glc-β1,2-Glu Glc-β1,2-Glu Rebaudioside AGlc-β1,2-Glu (β1,3-Glu) Glc- Stevioside Glc-β1,2-Glu Glc- RubusosideGlc- Glc- Steviol H Glc- monoglucosyl ester Rebaudioside B Glc-β1,2-Glu(β1,3-Glu) H Steviolbioside Glc-β1,2-Glu H Steviolmonoside Glc- HSteviol H H

In the table shown above, “Glc” designates glucose, and “Glc-β1,2-Glu”designates the inclusion of a β-1,2-glucoside bond. “Glc-β1,2-Glu(β1,3-Glu)” designates the inclusion of a β-1,2-glucoside bond andβ-1,3-glucoside bond.

As used herein, the term “steviol glycoside having at least oneunbranched β-1,2-glucoside bond” refers to a steviol glycosiderepresented by the above general formula (I) wherein at least one of thegroups R₁ and R₂ has an unbranched structure. An example thereof is asteviol glycoside selected from rebaudioside D, rebaudioside E,stevioside, and steviolbioside.

The method for producing a steviol glycoside according to the presentinvention cleaves the unbranched β-1,2-glucoside bond, thereby producinganother steviol glycoside.

The steviol glycoside obtained by the present method (hereinafterreferred to as “the steviol glycoside of the present invention”) variesdepending on the starting material, the “steviol glycoside having atleast one unbranched β-1,2-glucoside bond”. Examples thereof are shownbelow.

TABLE 2 Steviol Glycoside of Starting Material Present InventionRebaudioside D Rebaudioside A Rebaudioside E Stevioside, RubusosideStevioside Rubusoside Steviolbioside Steviolmonoside

In the method for producing a steviol glycoside according to the presentinvention, the steviol glycoside having at least one unbranchedβ-1,2-glucoside bond for use as the starting material can be obtained byextraction from Stevia rebaudiana or Rubus suavissimus followed bypurification using known methods including extraction with anappropriate solvent (an aqueous solvent such as water, or an organicsolvent such as an alcohol, ether, or acetone), a gradient between waterand ethyl acetate or other organic solvent, high performance liquidchromatography (HPLC), gas chromatography, time-of-flight massspectrometry (TOF-MS), and ultra (high) performance liquidchromatography (UPLC). Alternatively, a commercially-available productmay be used as the steviol glycoside having at least one unbranchedβ-1,2-glucoside bond for use as the starting material.

In some embodiments of the present invention, an unbranchedβ-1,2-glucoside bond of a steviol glycoside selected from stevioside,rebaudioside D, and steviolbioside is cleaved to produce a steviolglycoside selected from rubusoside, rebaudioside A, and steviolmonoside.In another embodiment, an unbranched β-1,2-glucoside bond ofrebaudioside E is cleaved to produce rubusoside.

The method for producing a steviol glycoside according to the presentinvention comprises reacting the protein of the present invention with asteviol glycoside having at least one unbranched β-1,2-glucoside bond,thereby cleaving said unbranched β-1,2-glucoside bond. The method of thepresent invention may further comprise purifying the steviol glycosideproduced in the above step.

The steviol glycoside produced in the above step can be purified usingknown methods including extraction with an appropriate solvent (anaqueous solvent such as water, or an organic solvent such as an alcohol,ether, or acetone), a gradient between water and ethyl acetate or otherorganic solvent, high performance liquid chromatography (HPLC), gaschromatography, time-of-flight mass spectrometry (TOF-MS), and ultra(high) performance liquid chromatography (UPLC).

A different steviol glycoside can be produced by combining the proteinof the present invention with another protein having a differentactivity.

Thus, in another embodiment, there is provided a method for producing atleast one of rebaudioside B and steviol comprising reacting the proteinof the present invention, a protein selected from the group consistingof proteins (d) to (f) shown below (hereinafter referred to as “thesecond active protein”), and a steviol glycoside having at least oneunbranched β-1,2-glucoside bond:

(d) a protein consisting of the amino acid sequence of SEQ ID NO: 7 or8;

(e) a protein consisting of an amino acid sequence wherein 1 to 77 aminoacids have been deleted, substituted, inserted, and/or added in theamino acid sequence of SEQ ID NO: 7 or 8, and having an activity tocleave a monoglucoside bond and/or monoglucosyl ester bond of a steviolglycoside; and

(f) a protein having an amino acid sequence having 90% or more sequenceidentity to the amino acid sequence of SEQ ID NO: 7 or 8, and having anactivity to cleave a monoglucoside bond and/or monoglucosyl ester bondof a steviol glycoside.

In this method, the steviol glycoside having at least one unbranchedβ-1,2-glucoside bond may comprise one or more steviol glycosidesselected from rebaudioside A, rebaudioside D, stevioside, rubusoside,steviolmonoside, and steviol monoglucosyl ester.

As used herein, the phrase “activity to cleave a monoglucoside bondand/or monoglucosyl ester bond of a steviol glycoside” refers to amonoglucoside bond and/or monoglucosyl ester bond in a steviolglycoside, which is a glycoside wherein glucose is linked to theaglycone, steviol. In an embodiment, the bond to be cleaved is, but notlimited to, a monoglucoside bond at the 13 position. In anotherembodiment, the bond to be cleaved is, but not limited to, amonoglucosyl ester bond at the 19 position.

The activity to cleave a monoglucoside bond and/or monoglucosyl esterbond of a steviol glycoside can be confirmed by reacting the secondactive protein with a steviol glycoside (rubusoside, for example) havingeither a monoglucoside bond or monoglucosyl ester bond or both of thebonds, purifying the resulting reaction product, and analyzing thepurified product using a known technique such as liquid chromatography(LC).

The phrase “amino acid sequence wherein 1 to 77 amino acid residues havebeen deleted, substituted, inserted, and/or added in the amino acidsequence of SEQ ID NO: 7 or 8” means that 1 to 77 amino acid residueshave been deleted, substituted, inserted, and/or added at any 1 to 77positions in the same sequence, wherein two or more of deletion,substitution, insertion, and addition may occur simultaneously.

Examples of amino acid residues that are interchangeable are aspreviously described.

The second active protein may not contain any secretory signal peptideor may contain a secretory signal peptide. When the protein of thepresent invention contains a secretory signal peptide, it preferablycontains the secretory signal peptide at its N-terminus. The secretorysignal peptide refers to a peptide domain that serves to causeextracellular secretion of a protein bound to the secretory signalpeptide. Amino acid sequences of such secretory signal peptides andpolynucleotide sequences encoding such amino acid sequences have beenwell known and reported in the art.

The second active protein can be obtained by, for example, expressing apolynucleotide encoding this protein (such as a polynucleotideconsisting of SEQ ID NO: 5 or 6) in appropriate host cells, although itcan also be produced by a chemical synthesis method such as the Fmocmethod (fluorenylmethyloxycarbonyl method) or the tBoc method(t-butyloxycarbonyl method). The second active protein can also bechemically synthesized using a peptide synthesizer from AAPPTec LLC,Perkin Elmer Inc., Protein Technologies Inc., PerSeptive Biosystems,Applied Biosystems, or SHIMADZU CORPORATION, for example.

The “steviol glycoside” is as previously described. Examples of thesteviol glycoside having a monoglucoside bond and/or monoglucosyl esterbond include rebaudioside A, stevioside, rubusoside, steviolmonoside,and steviol monoglucosyl ester.

The second active protein cleaves the monoglucoside bond and/ormonoglucosyl ester bond. The resulting product varies depending on thestarting material, the “steviol glycoside having at least onemonoglucoside bond and/or monoglucosyl ester bond”, as follows. Examplesthereof are shown below.

TABLE 3 Starting Material Product Rebaudioside A Rebaudioside BStevioside Steviolbioside Rubusoside Steviol Steviolmonoside Steviolmonoglucosyl ester

With the method for producing at least one of rebaudioside B and steviolcomprising reacting the protein of the present invention, the secondactive protein, and one or more steviol glycosides selected fromrebaudioside A, rebaudioside D, stevioside, rubusoside, steviolmonoside,and steviol monoglucosyl ester, based on the activity of the secondactive protein, rebaudioside B and steviol can be obtained as productsfrom rebaudioside A, rebaudioside D, and stevioside as shown below.

TABLE 4 Starting Material Product Rebaudioside D Rebaudioside BRebaudioside A Stevioside Steviol Rubusoside Steviolmonoside Steviolmonoglucosyl ester

2. Method for Producing the Steviol Glycoside of the Present InventionUsing a Non-Human Transformant

The protein of the present invention is a koji mold-derived secretoryenzyme or a variant thereof, and is expected to have high activity in anextracellular environment. In this case, the steviol glycoside of thepresent invention can be produced by introducing a polynucleotideencoding the protein of the present invention (see “the polynucleotideof the present invention” described below) into host cells derived frombacteria, fungi, plants, insects, non-human mammals, or the like, forextracellular expression of the protein of the present invention, and byreacting the protein of the present invention with a steviol glycosidehaving a β-1,2-glucoside bond. Alternatively, depending on the host, thesteviol glycoside of the present invention can be produced by expressingthe protein of the present invention in the host cells.

Thus, the present invention provides a method for producing a steviolglycoside having no unbranched β-1,2-glucoside bond comprising culturinga non-human transformant (hereinafter referred to as “the transformantof the present invention”) obtained by introducing a polynucleotideselected from the group consisting of polynucleotides (a) to (e) shownbelow (hereinafter referred to as “the polynucleotide of the presentinvention”) into a host producing a steviol glycoside having at leastone unbranched β-1,2-glucoside bond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1;

(b) a polynucleotide encoding a protein consisting of the amino acidsequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acidsequence wherein 1 to 77 amino acids have been deleted, substituted,inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4,and having an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequencehaving 90% or more sequence identity to the amino acid sequence of SEQID NO: 3 or 4, and having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditionsto a polynucleotide consisting of a nucleotide sequence complementary toa polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1and which encodes a protein having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside.

As used herein, the term “polynucleotide” refers to DNA or RNA.

Examples of the polynucleotide encoding a protein consisting of theamino acid sequence of SEQ ID NO: 3 or 4 include a polynucleotideconsisting of the nucleotide sequence of SEQ ID NO: 1. Examples of thepolynucleotide encoding a protein consisting of the amino acid sequenceof SEQ ID NO: 8 include a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 5.

Examples of the “protein consisting of an amino acid sequence wherein 1to 77 amino acids have been deleted, substituted, inserted, and/or addedin the amino acid sequence of SEQ ID NO: 3 or 4, and having an activityto cleave an unbranched β-1,2-glucoside bond of a steviol glycoside” areas described above.

Examples of the “protein having an amino acid sequence having 90% ormore sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4,and having an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside” are as described above.

As used herein, the phrase “a polynucleotide which hybridizes underhighly stringent conditions” refers to a polynucleotide obtained bymeans of a hybridization method such as colony hybridization, plaquehybridization, or Southern hybridization, using, as a probe, all of or aportion of a polynucleotide consisting of a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 1 or apolynucleotide consisting of a nucleotide sequence complementary to anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or4. For hybridization, methods as described in “Sambrook & Russell,Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor,Laboratory Press 2012” and “Ausubel, Current Protocols in MolecularBiology, John Wiley & Sons 1987-1997”, for example, can be used.

As used herein, the term “highly stringent conditions” refers to, forexample, the following conditions: 5×SSC, 5×Denhardt's solution, 0.5%SDS, 50% formamide, 50° C.; 0.2×SSC, 0.1% SDS, 60° C.; 0.2×SSC, 0.1%SDS, 62° C.; or 0.2×SSC, 0.1% SDS, 65° C.; although not limited thereto.Under these conditions, it is expected that DNA having a higher sequenceidentity will be efficiently obtained at a higher temperature. Note,however, that a plurality of factors such as temperature, probeconcentration, probe length, ionic strength, time, and saltconcentration are considered to affect the stringency of hybridization,and a person skilled in the art will be able to achieve the samestringency by selecting these factors as appropriate.

When a commercially available kit is used for hybridization, the AlkphosDirect Labelling and Detection System (GE Healthcare), for example, canbe used. In this case, hybridization is accomplished in accordance withthe protocol attached to the kit, i.e., a membrane may be incubatedovernight with a labeled probe and then washed with a primary washingbuffer containing 0.1% (w/v) SDS at 55 to 60° C. to detect thehybridized DNA. Alternatively, when a commercially available reagent(e.g., PCR labeling mix (Roche Diagnostics)) is used for digoxigenin(DIG) labeling of a probe during probe preparation based on all of or aportion of a nucleotide sequence complementary to the nucleotidesequence of SEQ ID NO: 1 of a nucleotide sequence complementary to anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 or4, the DIG nucleic acid detection kit (Roche Diagnostics) may be usedfor detection of hybridization.

In addition to those described above, examples of other hybridizablepolynucleotides include DNA sharing 60% or more, 61% or more, 62% ormore, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more,68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% ormore, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more,79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% ormore, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more,90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% ormore, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more,99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% ormore, 99.7% or more, 99.8% or more, or 99.9% or more sequence identitywith DNA of the nucleotide sequence of SEQ ID NO: 1 or DNA encoding theamino acid sequence of SEQ ID NO: 3 or 4, as calculated by the homologysearch software BLAST using default parameters.

Note that the sequence identity of amino acid sequences or nucleotidesequences can be determined using the BLAST algorithm developed byKarlin and Altschul (Basic Local Alignment Search Tool) (Proc. Natl.Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873,1993). When BLAST is used, default parameters in each program are used.

The polynucleotide of the present invention may further contain apolynucleotide consisting of a nucleotide sequence encoding a secretorysignal peptide. Preferably, the polynucleotide of the present inventioncontains, at its 5′ end, the polynucleotide consisting of a nucleotidesequence encoding a secretory signal peptide. The secretory signalpeptide is as described above. Such a secretory signal peptide can beselected as appropriate, depending on the host into which thepolynucleotide of the present invention is to be introduced. Forexample, when the host is yeast, examples of secretory signal peptidesinclude yeast-derived secretory signal peptides, such as MF(ALPHA)1signal peptide, PHO5 signal peptide, and SUC2 signal peptide. Examplesof polynucleotides encoding MF(ALPHA)1 signal peptide, PHO5 signalpeptide, and SUC2 signal peptide include polynucleotides consisting ofthe nucleotide sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, and SEQID NO: 35, respectively. The amino acid sequences of MF(ALPHA)1 signalpeptide, PHO5 signal peptide, and SUC2 signal peptide are shown in SEQID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 36, respectively. When the hostis koji mold, examples of secretory signal peptides include kojimold-derived signal peptides, such as a peptide consisting of the aminoacid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. The polynucleotideencoding the peptide consisting of the amino acid sequence of SEQ ID NO:28 or SEQ ID NO: 30 is a polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 27 or SEQ ID NO: 29, respectively, for example.

The above-described polynucleotide of the present invention can beobtained using a known genetic engineering technique or a knownsynthesis technique.

The polynucleotide of the present invention is preferably inserted intoan appropriate expression vector for introduction into a host.

An appropriate expression vector is typically configured to include:

(i) a promoter transcribable in host cells;

(ii) the polynucleotide of the present invention ligated to thepromoter; and

(iii) an expression cassette containing, as constituent elements,signals that function in the host cells for transcription terminationand polyadenylation of an RNA molecule.

Examples of methods for preparing such an expression vector include,although not particularly limited to, using plasmids, phages, cosmids,or the like.

The specific type of the vector is not particularly limited, and anyvector expressible in host cells may be selected as appropriate.Specifically, an appropriate promoter sequence may be selected inaccordance with the type of the host cells to ensure the expression ofthe polynucleotide of the present invention, and this promoter sequenceand the polynucleotide of the present invention may then be integratedinto any of various plasmids, for example, for use as an expressionvector.

The expression vector of the present invention contains an expressioncontrol region (e.g., a promoter, a terminator, and/or a replicationorigin), depending on the type of the host into which the expressionvector is to be introduced. For bacterial expression vectors, commonlyused promoters (e.g., trc promoter, tac promoter, and lac promoter) areused. Examples of yeast promoters include glyceraldehyde-3-phosphatedehydrogenase promoter and PHO5 promoter. Examples of filamentous fungipromoters include amylase and trpC. Moreover, examples of promoters forexpression of a target gene in plant cells include cauliflower mosaicvirus 35S RNA promoter, rd29A gene promoter, rbcS promoter, and mac-1promoter configured to have the enhancer sequence of the above-mentionedcauliflower mosaic virus 35S RNA promoter at the 5′-side ofAgrobacterium-derived mannopine synthase promoter sequence. Examples ofpromoters for animal cell hosts include viral promoters (e.g., SV40early promoter and SV40 late promoter). Examples of promoters induciblyactivated by external stimulation include mouse mammary tumor virus(MMTV) promoter, tetracycline-responsive promoter, metallothioneinpromoter, and heat-shock protein promoter.

The expression vector preferably contains at least one selection marker.For use as such a marker, auxotrophic markers (ura5, niaD), drugresistance markers (hygromycin, zeocin), geneticin resistance gene(G418r), copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad.Sci. USA, vol. 81, p. 337, 1984), cerulenin resistance genes (fas2m,PDR4) (Junji Inokoshi et al., Biochemistry, vol. 64, p. 660, 1992;Hussain et al., Gene, vol. 101, p. 149, 1991), and the like areavailable.

While the method for preparing (producing) the transformant of thepresent invention is not particularly limited, the transformant of thepresent invention may be prepared by, for example, introducing anexpression vector containing the polynucleotide of the present inventioninto a host to transform the host. The host to be used herein is notparticularly limited as long as it produces a steviol glycoside havingat least one unbranched β-1,2-glucoside bond, and may include not only aplant such as Stevia rebaudiana that produces a steviol glycoside havingat least one unbranched β-1,2-glucoside bond, but also a host obtainedby introducing a gene required for the production of a steviol glycosidehaving at least one unbranched β-1,2-glucoside bond into cells or anorganism that does not originally produce a steviol glycoside having atleast one unbranched β-1,2-glucoside bond. Examples of the “generequired for the production of a steviol glycoside having at least oneunbranched β-1,2-glucoside bond” include genes having steviol or steviolglycoside synthesis activity such as those described in WO 2011/093509.Any of conventionally known various types of cells or organisms can besuitably used as the cells or organism to be transformed. Examples ofthe cells to be transformed include bacteria such as Escherichia coli,yeast (budding yeast Saccharomyces cerevisiae, fission yeastSchizosaccharomyces pombe), filamentous fungi (koji mold Aspergillusoryzae, Aspergillus sojae), plant cells, and non-human animal cells.Appropriate media and conditions for culturing the above-described hostcells are well known in the art. Likewise, the organism to betransformed is not particularly limited, and examples include variousmicroorganisms, plants, and non-human animals described above asexamples of host cells. The transformant is preferably yeast or a plant.

For transformation of the host cells, commonly used known methods can beused. For example, transformation can be accomplished usingelectroporation (Mackenxie, D. A. et al., Appl. Environ. Microbiol.,vol. 66, p. 4655-4661, 2000), the particle delivery method (described inJP 2005-287403 A entitled “Breeding Method of Lipid Producing Fungi”),the spheroplast method (Proc. Natl. Acad. Sci. USA, vol. 75, p. 1929,1978), the lithium acetate method (J. Bacteriology, vol. 153, p. 163,1983), and other methods as described in Methods in yeast genetics, 2000Edition: A Cold Spring Harbor Laboratory Course Manual, although notlimited thereto. When a gene is introduced into a plant or into tissuesor cells derived from a plant, a method selected from the Agrobacteriummethod (Plant Molecular Biology Manual, Gelvin, S. B. et al., AcademicPress Publishers), particle gun method, PEG method, electroporation,etc. can be used as appropriate.

For other standard molecular biological techniques, reference may bemade to “Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol.4, Cold Spring Harbor Laboratory Press 2012” and “Methods in YeastGenetics, A laboratory manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.)”, for example.

When the transformant is yeast or koji mold, the transformant isobtained by introducing a recombinant vector containing thepolynucleotide of the present invention into yeast or koji mold suchthat a polypeptide encoded by the polynucleotide can be expressed. Theyeast or koji mold transformed with the polynucleotide of the presentinvention expresses a higher level of the protein of the presentinvention than in the wild-type counterpart. Thus, the expressed proteinof the present invention reacts with the steviol glycoside having atleast one unbranched β-1,2-glucoside bond produced in the yeast or kojimold, thereby cleaving said unbranched β-1,2-glucoside bond. As aresult, the steviol glycoside of the present invention having nounbranched β-1,2-glucoside bond is produced in the cells or culturemedium of the yeast or koji mold, preferably in the culture medium.

When the transformant is a plant, the transformant is obtained byintroducing a recombinant vector containing the polynucleotide of thepresent invention into a plant such that a protein encoded by thepolynucleotide can be expressed. The plant to be transformed in thepresent invention refers to any of whole plants, plant organs (e.g.,leaves, petals, stems, roots, and seeds), plant tissues (e.g.,epidermis, phloem, parenchyma, xylem, vascular bundles, palisade tissue,and spongy parenchyma) or plant cultured cells, or various forms ofplant cells (e.g., suspension cultured cells), protoplasts, leafsections, calli, and the like. The plant used for transformation is notparticularly limited as long as it is a plant that produces a steviolglycoside having at least one unbranched β-1,2-glucoside bond, or aplant that does not originally produce a steviol glycoside having atleast one unbranched β-1,2-glucoside bond, but can produce a steviolglycoside having at least one unbranched β-1,2-glucoside bond throughthe introduction of a required gene. The plant used for transformationmay be a plant in the class of either monocotyledons or dicotyledons.The introduction of the polynucleotide of the present invention into theplant can be confirmed by using PCR, Southern hybridization, or Northernhybridization, for example. Once a transformed plant in which thepolynucleotide of the present invention has been integrated into thegenome is obtained, progeny plants can be produced by sexual or asexualreproduction of the plant. Moreover, seeds, fruits, cuttings, tubers,root tubers, rootstocks, calli, protoplasts or the like can be obtainedfrom this plant or progeny plants thereof, or clones thereof, and usedto achieve mass production of the plant. The plant transformed with thepolynucleotide of the present invention (hereinafter, “the plant of thepresent invention”) contains a greater amount of the protein of thepresent invention than in the wild-type counterpart. Thus, the proteinof the present invention reacts with the steviol glycoside having atleast one unbranched β-1,2-glucoside bond produced in the plant of thepresent invention, thereby cleaving said unbranched β-1,2-glucosidebond. As a result, the steviol glycoside of the present invention havingno β-1,2-glucoside bond is produced in the plant.

The transformant in some embodiments of the present invention or theculture medium thereof has a content of the Steviol glycoside of thepresent invention higher than that in the wild-type counterpart, and anextract or the culture medium of the transformant contains a highconcentration of the Steviol glycoside of the present invention. Anextract of the transformant of the present invention can be obtained byhomogenating the transformant with glass beads, a homogenizer, or asonicator, for example, centrifuging the homogenate, and collecting thesupernatant. When the Steviol glycoside of the present inventionaccumulates in the culture medium, the transformant and the culturesupernatant may be separated using a standard method (e.g.,centrifugation or filtration) after the completion of culture, therebyobtaining the culture supernatant containing the Steviol glycoside ofthe present invention.

The extract or culture supernatant thus obtained may be furthersubjected to a purification step. The Steviol glycoside of the presentinvention may be purified in accordance with a standard separation andpurification method. Specific methods for purification are the same asdescribed above.

3. Method for Preparing the Steviol Glycoside of the Present InventionUsing an Enzyme Agent Derived from Non-Human Transformed Cells

The steviol glycoside of the present invention can be produced by usingan enzyme agent derived from transformed cells expressing the protein ofthe present invention, which are obtained by introducing thepolynucleotide of the present invention into host cells derived frombacteria, fungi, plants, insects, non-human mammals, or the like, forexpression of the protein of the present invention, i.e., by contactingthe enzyme agent derived from transformed cells expressing the proteinof the present invention with a steviol glycoside having at least oneunbranched β-1,2-glucoside bond. The “enzyme agent derived fromtransformed cells” is not limited as long as it is prepared usingtransformed cells, and contains the protein of the present invention.Examples of the enzyme agent include transformed cells themselves, atransformed cell homogenate itself, a transformed cell culturesupernatant itself, and a purified product thereof. Thus, the presentinvention provides a method for producing a steviol glycoside comprisingcontacting an enzyme agent derived from a non-human transformed cellobtained by introducing, into a host cell, a polynucleotide selectedfrom the group consisting of polynucleotides (a) to (e) shown below,with a steviol glycoside having at least one unbranched β-1,2-glucosidebond, thereby cleaving said unbranched β-1,2-glucoside bond:

(a) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO:1;

(b) a polynucleotide encoding a protein consisting of the amino acidsequence of SEQ ID NO: 3 or 4;

(c) a polynucleotide encoding a protein consisting of an amino acidsequence wherein 1 to 77 amino acids have been deleted, substituted,inserted, and/or added in the amino acid sequence of SEQ ID NO: 3 or 4,and having an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside;

(d) a polynucleotide encoding a protein having an amino acid sequencehaving 90% or more sequence identity to the amino acid sequence of SEQID NO: 3 or 4, and having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside; and

(e) a polynucleotide which hybridizes under highly stringent conditionsto a polynucleotide consisting of a nucleotide sequence complementary toa polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1and which encodes a protein having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside.

The polynucleotide selected from the group consisting of polynucleotides(a) to (e) shown above is the polynucleotide of the present invention,which is the same as described above.

The polynucleotide of the present invention may further contain apolynucleotide consisting of a nucleotide sequence encoding a secretorysignal peptide. Preferably, the polynucleotide of the present inventioncontains, at its 5′ end, the polynucleotide consisting of a nucleotidesequence encoding a secretory signal peptide. The secretory signalpeptide and the polynucleotide consisting of a nucleotide sequenceencoding the secretory signal peptide are the same as described above.

The polynucleotide of the present invention is preferably inserted intoan appropriate expression vector for introduction into host cells. Anappropriate expression vector is the same as described above.

While the method for preparing the transformed cells of the presentinvention is not particularly limited, the transformed cells of thepresent invention may be prepared by, for example, introducing anexpression vector containing the polynucleotide of the present inventioninto host cells to transform the host cells. The cells to be transformedare the same as described above. The method for transforming the hostcells is as described above.

The transformed cells of the present invention are obtained by, forexample, introducing a recombinant vector containing the polynucleotideof the present invention into the host cells such that a polypeptideencoded by the polynucleotide can be expressed. The host cellstransformed with the polynucleotide of the present invention express ahigher level of the protein of the present invention than in thewild-type counterpart. Thus, the steviol glycoside of the presentinvention can be obtained by using an enzyme agent derived fromtransformed cells expressing the protein of the present invention, i.e.,by contacting the enzyme agent derived from transformed cells expressingthe protein of the present invention with a steviol glycoside having atleast one unbranched β-1,2-glucoside bond.

The term “contact” refers to causing the enzyme agent derived from thetransformed cells of the present invention and the steviol glycosidehaving at least one unbranched β-1,2-glucoside bond to exist in the samereaction or culture system. The term “contact” includes, for example,adding the steviol glycoside having at least one unbranchedβ-1,2-glucoside bond to a container containing the enzyme agent derivedfrom the transformed cells of the present invention, mixing the enzymeagent derived from the transformed cells of the present invention andthe steviol glycoside having at least one unbranched β-1,2-glucosidebond, and adding the enzyme agent derived from the transformed cells ofthe present invention to a container containing the steviol glycosidehaving at least one unbranched β-1,2-glucoside bond.

The terms “steviol glycoside” and “steviol glycoside having at least oneunbranched β-1,2-glucoside bond” are the same as described above.

The Steviol glycoside of the present invention thus obtained can be usedfor such purposes as the production of foods, sweeteners, flavors,pharmaceutical products, and industrial raw materials (raw materials forcosmetics, soaps, and the like), for example, in accordance withconventional methods.

Examples of foods include nutritional supplements, health foods,functional foods, foods for children, and foods for the elderly. As usedherein, the term “foods” refers collectively to edible materials in theform of solids, fluids, liquids, and mixtures thereof.

Note that all documents, as well as laid-open application publications,patent application publications, and other patent documents cited hereinshall be incorporated herein by reference.

Examples

The present invention will be more specifically described hereinafterwith reference to examples, which are not intended to limit the scope ofthe present invention.

1. Cloning of Steviol-Hydrolyzing Enzyme Genes

1-(1) Genes

AO090009000356 and AO090701000274 were cloned and expressed in yeast toexamine the activity.

1-(2) Cloning of cDNAs of Koji Mold

Koji mold Aspergillus oryzae var. Brunneus (IFO30102) was inoculated toa GY plate (2% glucose, 0.5% yeast extract, and 2% agar), and culturedat 25° C. for 3 days. The grown cells were collected from the GY plate,and total RNA was extracted using RNeasy (QIAGEN). A cDNA wassynthesized using the SuperScript Double-Stranded cDNA Synthesis Kit(Life Technologies).

The following primers were designed based on the DNA sequence ofAO090009000356 and AO090701000274:

AO090009000356 (hereinafter expressed as AOBGL1) AOBGL1-1:(SEQ ID NO: 9) 5′-AGATCTATGAAGCTTGGTTGGATCGAGGT-3′ AOBGL1-2:(SEQ ID NO: 10) 5′-GTCGACTTACTGGGCCTTAGGCAGCGA-3′AO090701000274 (hereinafter expressed as AOBGL3) AOBGL3-1:(SEQ ID NO: 11) 5′-GCGGCCGCatggtttccggtgtctttacgaagg-3′ AOBGL3-2:(SEQ ID NO: 12) 5′-GGATCCtcactgcacatagaaagtagcattgcc-3′

Approximately 2.6 kbp or 2.3 kbp of a DNA fragment amplified by PCRusing ExTaq (Takara Bio), using each of the cDNAs synthesized asdescribed above as a template, was cloned using the TOPO-TA cloning Kit(Life Technologies). Each of the plasmids obtained herein was designatedas pCR-AOBGL1 or pCR-AOBGL3.

2. Expression in Yeast

2-(1) Construction of Yeast Expression Vectors and Acquisition ofTransformed Strains

A DNA fragment obtained by digesting the yeast expression vector pYE22m(Biosci. Biotech. Biochem., 59, 1221-1228, 1995) with restrictionenzymes BamHI and SalI and approximately 2.6 kbp of a DNA fragmentobtained by digesting pCR-AOBGL1 with restriction enzymes BglII and SalIwere ligated using the DNA Ligation Kit Ver.1 (Takara Bio), and theresulting plasmid was designated as pYE-AOBGL1. Additionally, a DNAfragment obtained by digesting pYE22 mN with restriction enzymes NotIand BamHI and approximately 2.3 kbp of a DNA fragment obtained bydigesting pCR-AOBGL3 with restriction enzymes NotI and BamHI wereligated in the same manner as above, and the resulting plasmid wasdesignated as pYE-AOBGL3. S. cerevisiae strain EH13-15 (trp1, MATα)(Appl. Microbiol. Biotechnol., 30, 515-520, 1989) was used as theparental strain for transformation.

Each of the plasmids pYE22m (control), pYE-AOBGL1 (for expression ofAOBGL1), and pYE-AOBGL3 (for expression of AOBGL3) was used to transformstrain EH13-15 in accordance with the lithium acetate method. A strainthat grew on SC-Trp (containing, per liter, 6.7 g of Yeast nitrogen basew/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acid powder(a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 g ofaspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g ofleucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine,11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine,and 0.6 g of uracil) agar medium (2% agar) was selected as thetransformed strain.

The selected strain was applied to SC-Trp agar medium containing 0.004%of X-β-Glc, and cultured at 30° C. for 3 days. As a result, neither thestrain transformed with any of the plasmids pYE-AOBGL1 and pYE-AOBGL3nor the strain transformed with the control pYE22m was stained blue, andno X-β-Glc degrading activity was confirmed.

Meanwhile, one platinum loop of the selected strain was inoculated to 10mL of SC-Trp liquid medium supplemented with 1/10 volume of 1 Mpotassium phosphate buffer, and cultured with shaking at 30° C. and 125rpm for 2 days. The resulting culture was separated into the culturesupernatant and cells by centrifugation. The cells were suspended in 50mM sodium phosphate buffer (pH 7.0) containing 0.1% CHAPS solution andthen homogenated with glass beads, and the supernatant obtained bycentrifugation was used as the cell homogenate. The obtained culturesupernatant or cell homogenate was examined for its pNP-β-Glc activity.

As a result, both the culture supernatant and cell homogenate for eachtype of transformed strain including the control exhibited pNP-β-Glcdegrading activity, and no significant difference in activity wasobserved between them.

These results suggested that the introduction of the plasmid pYE-AOBGL1or pYE-AOBGL3 into yeast strain EH13-15 does not allow expression of anactivated protein having β-glucosidase activity. Moreover, the need forthe deletion of an endogenous gene responsible for β-glucosidaseactivity in yeast was indicated.

2-(2) Creation of ΔExg1 ΔExg2 Yeast Host Strains

A strain with deletion of the EXG1 (YLR300w) gene considered to beresponsible for most of the extracellular β-glucosidase activity inyeast and its homolog EXG2 (YDR261c) gene was used as the host strainfor transformation. This host strain was created as follows:

Each of Δexg1 strain (MATalpha his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0; clone ID:15210; Open Bio Systems) and Δexg2 strain (MATa his3Δ1 leu2Δ0 met15Δ0ura3Δ0; clone ID: 3620; Open BioSystems) was applied to YPD agar medium,and cultured at 30° C. for 2 days. The cells of each strain were scrapedwith a platinum loop and mixed on SC-Met, Lys (containing, per liter,6.7 g of Yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose,and 1.3 g of amino acid powder (a mixture of 1.25 g of adenine sulfate,0.6 g of arginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g ofhistidine, 1.8 g of leucine, 1.5 g of phenylalanine, 11.25 g of serine,0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine, 1.2 g oftryptophan, and 0.6 g of uracil) agar medium (2% agar), and the mixturewas cultured at 30° C. for 2 days. The grown strain was considered to bea hetero-diploid obtained by hybridization of the two strains. Theobtained strain was applied to YPD agar medium and cultured at 30° C.for 2 days, and then the cells were scraped with a platinum loop,applied to 0.5% potassium acetate agar medium (2% agar), and cultured atroom temperature for 5 days, thus forming spores. Tetrad dissection wasperformed to separate haploid strains. Genotypes of the obtained strainswere confirmed by PCR, and Δexg1 Δexg2-1 strain (his3Δ1 leu2Δ0 lys2Δ0ura3Δ0) was selected.

Using the genomic DNA of yeast strain S288C, PCR was performed with thefollowing primers TRP1-F and TRP1-R, using KOD-Plus (Toyobo).Approximately 2.7 kbp of the amplified DNA fragment was cloned using theZero Blunt TOPO PCR cloning Kit (Life Technologies), thus obtaining aplasmid pCR-TRP1.

TRP1-F: (SEQ ID NO: 13) TACTATTAGCTGAATTGCCACTGCTATCG TRP1-R:(SEQ ID NO: 14) TCTACAACCGCTAAATGTTTTTGTTCG

2.7 kbp of a DNA fragment obtained by digesting pPRGINFRT3-103 (JapanesePatent Laid-Open No. 2001-120276) with restriction enzymes EcoRI andHindIII was blunt-ended using the Blunting Kit (Takara Bio), and thenligated to a DNA fragment obtained by digesting the plasmid pCR-TRP1with restriction enzymes HpaI and StuI, using Ligation High (Toyobo),thus obtaining a plasmid pCR-Δtrp1:URA3-FRT. Using this plasmid as atemplate, PCR was performed with the primers TRP1-F and TRP1-R, usingKOD-Plus (Toyobo). Then, 4.4 kbp of the resulting DNA fragment was usedto transform Δexg1 Δexg2-1 strain in accordance with the lithium acetatemethod, and a strain that grew on SC-Ura (containing, per liter, 6.7 gof Yeast nitrogen base w/o amino acids (DIFCO), 20 g of glucose, and 1.3g of amino acid powder (a mixture of 1.25 g of adenine sulfate, 0.6 g ofarginine, 3 g of aspartic acid, 3 g of glutamic acid, 0.6 g ofhistidine, 1.8 g of leucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 gof phenylalanine, 11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine,6 g of threonine, and 1.2 g of tryptophan) agar medium (2% agar) wasselected as the transformed strain. The transformed strain was culturedon YPGal medium (yeast extract: 2%, polypeptone: 1%, galactose: 2%) andthen applied to SC+5-FOA (containing, per liter, 6.7 g of Yeast nitrogenbase w/o amino acids (DIFCO), 20 g of glucose, and 1.3 g of amino acidpowder (a mixture of 1.25 g of adenine sulfate, 0.6 g of arginine, 3 gof aspartic acid, 3 g of glutamic acid, 0.6 g of histidine, 1.8 g ofleucine, 0.9 g of lysine, 0.6 g of methionine, 1.5 g of phenylalanine,11.25 g of serine, 0.9 g of tyrosine, 4.5 g of valine, 6 g of threonine,1.2 g of tryptophan, and 0.6 g of uracil) agar medium (2% agar), and agrown strain was obtained as Δexg1 Δexg2-2 strain and used as the hostfor the following transformation.

2-(3) Substitution of Secretory Signal Sequences and Expression in Yeast

Nineteen amino acids (SEQ ID NO: 28) at the N-terminus of AOBGL1p and 22amino acids (SEQ ID NO: 30) at the N-terminus of AOBGL3 are estimated tobe secretory signal sequences.

Thus, for secretion and expression of each of AOBGL1 and AOBGL3 inyeast, the estimated secretory signal sequence was substituted with asecretory signal sequence of a yeast secretory protein. Initially, thefollowing oligodeoxynucleotides were synthesized and annealed, and theninserted into the EcoRI site of the vector pYE22m, thus creatingpYE-PacNhe.

PacI-NheI-F: (SEQ ID NO: 15) 5′-AATTAATTAAGAGCTAGCG-3′ PacI-NheI-R:(SEQ ID NO: 16) 5′-TTAATTCTCGATCGCTTAA-3

Using the plasmid pCR-AOBGL1 or pCR-ASBGL1 as a template, PCR wasperformed with the following primers Bgl2-AOBGL1-F and AOBGL1-2, usingKOD-Plus (Toyobo). Approximately 2.5 kbp of a DNA fragment obtained bydigesting the PCR-amplified DNA fragment with restriction enzymes BglIIand SalI was inserted into the sites of restriction enzymes BamHI andSalI of the vector pYE-PacNhe, thus constructing a plasmidpYE-PN-AOBGL1.

Bgl2-AOBGL1-F: (SEQ ID NO: 17) 5′-TAAGATCTAAGGATGATCTCGCGTACTCCCC-3AOBGL1-2: (SEQ ID NO: 18) 5′-GTCCACTTACTGGGCCTTAGGCAGCGA-3′

Using the plasmid pCR-ASBGL3 as a template, PCR was performed with thefollowing primers Bam-ASBGL3-F and Sal-ASBGL3-R, using KOD-Plus(Toyobo). Approximately 2.3 kbp of a DNA fragment obtained by digestingthe PCR-amplified DNA fragment with restriction enzymes BamHI and SalIwas inserted into the sites of restriction enzymes BamHI and SalI of thevector pYE-PacNhe, thus constructing a plasmid pYE-PN-AOBGL3.

Bam-AOBGL3-F: (SEQ ID NO: 19) 5′-AAGGATCCCAAGATGAGAAGCCTCGCTACAAGG-3Sal-AOBGL3-R: (SEQ ID NO: 20) 5′-GGGTCGACTCACTGCACATAGAAAGTAGCATTGCC-3′

The primers shown below were designed to construct a plasmid forexpression of a protein in which the estimated secretory signal sequenceof AOBGL1 or AOBGL3 was substituted with the secretory signal sequenceMF(ALPHA)1 (YPL187W) (the sequence of positions 1 to 19 of the aminoacid sequence (SEQ ID NO: 32) shown in FIG. 2A), PHO5 (YBR093C) (thesequence of positions 1 to 17 of the amino acid sequence (SEQ ID NO: 34)shown in FIG. 2B), or SUC2 (YIL162W) (the sequence of positions 1 to 19of the amino acid sequence (SEQ ID NO: 36) shown in FIG. 2C) of a yeastsecretory protein.

ScPHO5-F: (SEQ ID NO: 21) 5′-TAAATGTTTAAATCTGTTCTTTATTCAATTTTAGCCGCTTCTTTCCCCAA TGCAG-3′ ScPHO5-R:(SEQ ID NO: 22) 5′- CTAGCTGCATTGGCCAAAGAACCCGCTAALATTGAATAAACAACAGATTTAAACATTTAT-3′ ScSUC2-F: (SEQ ID NO: 23) 5′-TAAATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTTTGCACCCAA AATATCTCCAG-3′ScSUC2-R: (SEQ ID NO: 24) 5′-TAAATGAGATTTCCTTCAATTTTTACTCCACTTTTATTCGCAGCATCCTC CGCATTAGCTG-3′ScMF1-F: (SEQ ID NO: 25) 5′-TAAATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTC CGCATTAGCTG-3′ScMF1-R: (SEQ ID NO: 26) 5′-CTAGCAGCTAATCCCGAGCATGCTCCGAATAAAACTGCAGTAAAAATTGA AGGAAATCTCATTTAAT-3′

The combination of ScPHO5-F and ScPHO5-R, the combination of ScSUC2-Fand ScSUC2-R, and the combination of ScMF1-F and ScMF1-R were eachannealed, and then ligated to the plasmid pYE-PN-AOBGL1 or pYE-PN-ASBGL1digested with restriction enzymes PacI and NheI, thus obtaining thefollowing plasmids:

pYE-PHO5s-AOBGL1 (for expression of PHO5s-AOBGL1)pYE-SUC2s-AOBGL1 (for expression of SUC2s-AOBGL1)pYE-MF1s-AOBGL1 (for expression of MF1s-AOBGL1)pYE-PHO5s-AOBGL3 (for expression of PHO5s-AOBGL3)pYE-SUC2s-AOBGL3 (for expression of SUC2s-AOBGL3)pYE-MF1s-AOBGL3 (for expression of MF1s-AOBGL3)

Δexg1 Δexg2-2 strain was transformed with these plasmids in accordancewith the lithium acetate method, and a strain that grew on SC-Trp agarmedium was selected as the transformed strain.

The obtained transformed strain was applied to SD-Trp agar mediumcontaining 0.004% of X-β-Glc, and cultured at 30° C. for 3 days. As aresult, the cells and surrounding regions were stained blue in thestrain transformed with pYE-PHO5s-AOBGL1, pYE-SUC2s-AOBGL1,pYE-MF1s-AOBGL1, pYE-PHO5s-ASBGL1, pYE-SUC2s-ASBGL1, or pYE-MF1s-ASBGL1,suggesting that these strains had X-β-Glc hydrolyzing activity.

Meanwhile, in the strain transformed with the plasmid for expression ofAOBGL3, the cells and surrounding regions were not stained blue, and theactivity to hydrolyze X-β-Glc was not confirmed.

In the strain transfected with a control vector, the cells andsurrounding regions were not stained blue, showing that the strain didnot have the activity to hydrolyze X-β-Glc.

One platinum loop of the obtained transformed strain was inoculated to aliquid medium obtained by mixing 10 mL of SD-Trp liquid medium and 1 mLof 1 M potassium phosphate buffer, and cultured with shaking at 30° C.for 2 days. The culture was separated into the cells and culturesupernatant by centrifugation. 50 μL of the culture supernatant, 50 μLof a 0.2 M sodium citrate buffer, 50 μL of a 20 mM aqueous pNP-βGlcsolution, and 50 μL of water were mixed, and the mixture was reacted at37° C. for 1 hour, after which the increase in absorbance at 405 nm dueto β-glucosidase activity was examined. When AOBGL1 was expressed, theβ-glucosidase activity was confirmed as shown in Table 5. AOBGL1 wasconsidered to be expressed at the highest level when substituted withthe MF1 signal sequence.

TABLE 5 β-glucosidase activity in culture medium (Increase in absorbanceat 405 nm; reaction for 1 hour) Plasmid Δ 405 nm pYE-MF1s-AOBGL1 0.508pYE-PHO5s-AOBGL1 0.37 pYE-SUC2s-AOBGL1 0.369 pYE22m 0

However, when AOBGL3 was expressed, the β-glucosidase activity was notconfirmed under the same conditions.

2-(4) Activity of Recombinant Enzyme on Steviol Glycoside

AOBGL1 was confirmed to have an activity to convert stevioside intorubusoside.

3. Expression of AOBGL3 in Koji Mold

3-(1) Construction of Koji Mold Expression Vector

A DNA fragment obtained by digesting a koji mold vector pUNA (NationalResearch Institute of Brewing) with a restriction enzyme SmaI, andapproximately 2.3 kbp of a DNA fragment obtained by digesting theplasmid pCR-AOBGL3 with restriction enzymes NotI and BamHI andblunt-ending the end using Blunting Kit (Takara Bio), were ligated toobtain a plasmid pUNA-AOBGL3.

3-(2) Transformation of Koji Mold

Koji mold was transformed as follows.

Aspergillus oryzae niaD300 strain (National Research Institute ofBrewing) was used as a host. The host strain was inoculated to a PDAplate and cultured at 30° C. for about 1 week. In order to obtain aconidial suspension, the resulting conidia were suspended by adding 0.1%tween 80 and 0.8% NaCl. The suspension was filtered through Miraclothand then centrifuged to collect the conidia. The conidia were thenwashed with 0.1% tween 80 and 0.8% NaCl and suspended in sterilizedwater.

The conidia were applied to a CD plate, and DNA was introduced into theconidia by the particle delivery method. This was performed usingPDS-1000/He (Bio-Rad), tungsten M-10 particles, and a 1100 psi rupturedisc at a distance of 3 cm. A strain that grew on a CD plate(containing, per liter, 6 g of NaNO₃, 0.52 g of KCl, 1.52 g of KH₂PO₄,10 g of glucose, 2 ml of IM MgSO₄, 1 ml of a trace element solution(containing, per liter, 1 g of FeSO₄.7H₂O, 8.8 g of ZnSO₄.7H₂O, 0.4 g ofCuSO₄.5H₂O, 0.1 g of NaB₄O₇.10H₂O, and 0.05 g of (NH₄)₆Mo₇O₂₄.4H₂O), and20 g of agar (pH 6.5)) was selected as the transformed strain.

3-(3) Preparation of Conidial Suspension

BGL3-1 stain or C-1 strain was inoculated to a CD plate and cultured at30° C. for 7 days to form conidia. In order to obtain a conidialsuspension, the conidia were suspended by adding 0.1% tween 80 and 0.8%NaCl. The suspension was filtered through Miracloth and then centrifugedto collect the conidia. The conidia were then washed with 0.1% tween 80and 0.8% NaCl and suspended in sterilized water to prepare a conidialsuspension.

3-(4) Production of AOBGL3 by Liquid Culture

Conidia of BGL3-1 strain or C-1 strain were inoculated to a liquidculture medium for enzyme production (containing, per liter, 100 g ofmaltose, 1 g of Bacto-tryptone, 5 g of yeast extract, 1 g of NaNO₃, 0.5g of K₂HPO₄, 0.5 g of MgSO₄.7H₂O, and 0.01 g of FeSO₄.7H₂O) and culturedwith shaking at 30° C. for 2 days. The medium was filtered throughMiracloth to remove the cells and further filtered through a membranefiltration system (IWAKI) to collect the supernatant. The supernatantwas then concentrated by ultrafiltration through Amicon Ultra-15 50 k(Merck), and the buffer was replaced with a 50 mM sodium phosphatebuffer (pH 7.0) containing 0.1% CHAPS to obtain a crude enzyme solution.

3-(5) Examination of Activity (Liquid Culture)

The activity on various substrates was examined. Both thepNP-β-Glc:BGL3-1 strain and C-1 strain were stained yellow, showing thatboth had the activity to hydrolyze pNP-β-Glc.

The X-β-Glc:BGL3-1 strain was stained blue, showing that it had theactivity to hydrolyze X-β-Glc. By contrast, the C-1 strain was notstained blue, which was considered to indicate that the C-1 strain didnot have the activity to hydrolyze X-β-Glc. The activity on X-β-Glc wasattributed to an AOBGL3 gene product.

3-(6) Activity to Hydrolyze Steviol Glycoside

An enzyme solution derived from BGL3-1 strain or C-1 strain (liquidculture or plate culture) was examined for the activity to hydrolyzesteviol glycosides (rubusoside) as follows.

Reaction Conditions

50 μg/ml of substrate, 20 μl of the enzyme solution, and a 50 mM sodiumcitrate buffer (pH 5.0) were mixed to a total volume of 100 μl, and themixture was reacted at 50° C. The reaction mixture was passed throughSep-Pak C18 (Waters) washed with acetonitrile and equilibrated withwater. The reaction product was subsequently washed with 20%acetonitrile and then eluted with 50% acetonitrile. The eluate wasevaporated to dryness with SpeedVac. The resulting product was dissolvedin 100 μL of 80% acetonitrile, and the solution was subjected to HPLC.

The conditions for HPLC were as follows:

Column: COSMOSIL 5C₁₈-AR-II 4.6 mm I.D.×250 mm (Nacalai Tesque)

Mobile phase: A; acetonitrile, B;

B conc. 70%→30% 40 min linear gradient

Flow rate: 1 ml/min

Temperature: 40° C.

Detection: UV 210 nm

In summary, the enzyme activity to hydrolyze the glucoside bond at the13 position of rubusoside and the glucosyl ester bond at the 19 positionof rubusoside was exhibited through expression of AOBGL3. In view of thefact that an intermediate, steviol monoglucosyl ester, was detected inaddition to steviol in the reaction with rubusoside, the AOBGL3 proteinwas found to be an enzyme which, although capable of hydrolyzing boththe glucoside bond at the 13 position of rubusoside and the glucosylester bond at the 19 position of rubusoside, preferentially hydrolyzesthe glucoside bond at the 13 position.

1. A method for producing a steviol glycoside having no unbranchedβ-1,2-glucoside bond comprising reacting a protein selected from thegroup consisting of proteins (a) to (c) shown below with a steviolglycoside having at least one unbranched β-1,2-glucoside bond, therebycleaving said unbranched β-1,2-glucoside bond: (a) a protein consistingof the amino acid sequence of SEQ ID NO: 3 or 4; (b) a proteinconsisting of an amino acid sequence wherein 1 to 77 amino acids havebeen deleted, substituted, inserted, and/or added in the amino acidsequence of SEQ ID NO: 3 or 4, and having an activity to cleave anunbranched β-1,2-glucoside bond of a steviol glycoside; and (c) aprotein having an amino acid sequence having 90% or more sequenceidentity to the amino acid sequence of SEQ ID NO: 3 or 4, and having anactivity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside.
 2. The method according to claim 1, wherein the proteinselected from the group consisting of the proteins (a) to (c) furthercomprises a secretory signal peptide.
 3. The method according to claim2, wherein the secretory signal peptide is a peptide consisting of theamino acid sequence set forth in any one of SEQ ID NOS: 28, 30, 32, 34,and
 36. 4. The method according to claim 3, wherein the steviolglycoside having at least one unbranched β-1,2-glucoside bond isselected from rebaudioside D, rebaudioside E, stevioside, andsteviolbioside.
 5. The method according to claim 4, wherein the steviolglycoside having no unbranched β-1,2-glucoside bond is selected fromrubusoside, rebaudioside A, and steviolmonoside.
 6. A method forproducing a steviol glycoside having no unbranched β-1,2-glucoside bondcomprising culturing a non-human transformant obtained by introducing apolynucleotide selected from the group consisting of polynucleotides (a)to (e) shown below into a host producing a steviol glycoside having atleast one unbranched β-1,2-glucoside bond: (a) a polynucleotideconsisting of the nucleotide sequence of SEQ ID NO: 1; (b) apolynucleotide encoding a protein consisting of the amino acid sequenceof SEQ ID NO: 3 or 4; (c) a polynucleotide encoding a protein consistingof an amino acid sequence wherein 1 to 77 amino acids have been deleted,substituted, inserted, and/or added in the amino acid sequence of SEQ IDNO: 3 or 4, and having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside; (d) a polynucleotideencoding a protein having an amino acid sequence having 90% or moresequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, andhaving an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside; and (e) a polynucleotide which hybridizes underhighly stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 1 and which encodes a protein havingan activity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside.
 7. The method according to claim 6, wherein thepolynucleotide selected from the group consisting of the polynucleotides(a) to (e) further comprises a polynucleotide consisting of a nucleotidesequence encoding a secretory signal peptide.
 8. The method according toclaim 7, wherein the polynucleotide consisting of a nucleotide sequenceencoding a secretory signal peptide is a polynucleotide consisting ofthe nucleotide sequence set forth in any one of SEQ ID NOS: 27, 29, 31,33, and
 35. 9. The method according to claim 6, wherein thepolynucleotide is inserted into an expression vector.
 10. The methodaccording to claim 6, wherein the transformant is transformed koji mold,transformed yeast, or a transformed plant.
 11. A method for producing asteviol glycoside having no unbranched β-1,2-glucoside bond comprisingcontacting an enzyme agent derived from a non-human transformed cellobtained by introducing, into a host cell, a polynucleotide selectedfrom the group consisting of polynucleotides (a) to (e) shown below,with a steviol glycoside having at least one unbranched β-1,2-glucosidebond, thereby cleaving said β-1,2-glucoside bond: (a) a polynucleotideconsisting of the nucleotide sequence of SEQ ID NO: 1; (b) apolynucleotide encoding a protein consisting of the amino acid sequenceof SEQ ID NO: 3 or 4; (c) a polynucleotide encoding a protein consistingof an amino acid sequence wherein 1 to 77 amino acids have been deleted,substituted, inserted, and/or added in the amino acid sequence of SEQ IDNO: 3 or 4, and having an activity to cleave an unbranchedβ-1,2-glucoside bond of a steviol glycoside; (d) a polynucleotideencoding a protein having an amino acid sequence having 90% or moresequence identity to the amino acid sequence of SEQ ID NO: 3 or 4, andhaving an activity to cleave an unbranched β-1,2-glucoside bond of asteviol glycoside; and (e) a polynucleotide which hybridizes underhighly stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to a polynucleotide consisting of thenucleotide sequence of SEQ ID NO: 1 and which encodes a protein havingan activity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside.
 12. The method according to claim 11, wherein thepolynucleotide selected from the group consisting of the polynucleotides(a) to (e) further comprises a polynucleotide consisting of a nucleotidesequence encoding a secretory signal peptide.
 13. The method accordingto claim 12, wherein the polynucleotide consisting of a nucleotidesequence encoding a secretory signal peptide is a polynucleotideconsisting of the nucleotide sequence set forth in any one of SEQ IDNOS: 27, 29, 31, 33, and
 35. 14. The method according to claim 11,wherein the polynucleotide is inserted into an expression vector. 15.The method according to of claim 11, wherein the transformed cell istransformed koji mold, a transformed bacterium, or transformed yeast.16. The method according to of claim 11, wherein the steviol glycosidehaving at least one unbranched β-1,2-glucoside bond is selected fromrebaudioside D, rebaudioside E, stevioside, and steviolbioside.
 17. Themethod according to claim 16, wherein the steviol glycoside having nounbranched β-1,2-glucoside bond is selected from rubusoside,rebaudioside A, and steviolmonoside.
 18. A method for producing at leastone of rebaudioside B and steviol comprising reacting a protein selectedfrom the group consisting of proteins (a) to (c) shown below, a proteinselected from the group consisting of proteins (d) to (f) shown below,and a steviol glycoside having at least one unbranched β-1,2-glucosidebond: (a) a protein consisting of the amino acid sequence of SEQ ID NO:3 or 4; (b) a protein consisting of an amino acid sequence wherein 1 to77 amino acids have been deleted, substituted, inserted, and/or added inthe amino acid sequence of SEQ ID NO: 3 or 4, and having an activity tocleave an unbranched β-1,2-glucoside bond of a steviol glycoside; (c) aprotein having an amino acid sequence having 90% or more sequenceidentity to the amino acid sequence of SEQ ID NO: 3 or 4, and having anactivity to cleave an unbranched β-1,2-glucoside bond of a steviolglycoside; (d) a protein consisting of the amino acid sequence of SEQ IDNO: 7 or 8; (e) a protein consisting of an amino acid sequence wherein 1to 77 amino acids have been deleted, substituted, inserted, and/or addedin the amino acid sequence of SEQ ID NO: 7 or 8, and having an activityto cleave a monoglucoside bond and/or monoglucosyl ester bond of asteviol glycoside; and (f) a protein having an amino acid sequencehaving 90% or more sequence identity to the amino acid sequence of SEQID NO: 7 or 8, and having an activity to cleave a monoglucoside bondand/or monoglucosyl ester bond of a steviol glycoside.
 19. The methodaccording to claim 18, wherein the steviol glycoside having at least oneunbranched β-1,2-glucoside bond comprises one or more steviol glycosidesselected from rebaudioside D, rebaudioside E, stevioside, andsteviolbio.